Research will be conducted to obtain a better understanding at the molecular level of the following aspects of vaccinia virus replication: 1) The virion-associated DNA-dependent RNA-polymerase which we have recently solubilized will be purified and characterized. It will then be utilized to study in vitro transcription of vaccinia DNA in order to identify other viral components which are necessary for the production of vaccinia mRNA. 2) The type of association of purified vaccinia DNA-binding proteins with DNA will be determined and the role of such proteins in transcription of viral genes and other processes will be determined. Studies will be conducted on the structure of viral protein-DNA complexes. Experiments will be conducted to define the role of the host cell nucleus in the replication of poxviruses. 3) When guidelines permit, various fragments of the vaccinia genome will be cloned utilizing recombinant DNA techniques in appropriate lambda phage vectors. Appropriate clones will then be utilized in a variety of studies relating to an understanding of the viral transcription process and genome organization; and as a potentially powerful method for the production of site specific mutants of the virus.